1. Field of the Invention
This invention relates to the immobilisation of enzymes, more particularly to the immobilisation of enzymes suCh as L-methionine gamma-lyase and L-lysine decarboxylase. The invention also relates to an electrosensor for assaying amino acids in solution, comprising a pH-responsive electrode in combination with an immobilised enzyme which degrades the aminoacid with an acCompanying change in the pH of the solution.
2. Prior Art
Immobilised enzymes and their role in biocatalytic reactions are of considerable technological importance. Carriers which have been used to immobilise enzymes include polymer latices. In most instanCes, the polymers concerned have been derived from monomers or comonomers containing, functional groups. For instance U.S. Pat No. 4,064,080 discloses latices of styrene polymers having terminal aminophenylthio groups for immobilising proteins; Bahadur et al, Makromol. Chem. (1985), 186, 1387 describe core-shell latices of poly(methyl methacrylate-co-acrylic acid) having carboxyl groups on the surface of the polymer particles, and the immobilisation of alpha-chymotrypsin on the surface of the latex particles by chemical bonding using a carbodiimide coupling agent; and Hoshino et al, Kobunshi Ronbunshu, (1985), 42 (5), 305 describe the use of hydrolysed styrene-N-hydroxymethyl acrylamide copolymer latices to immobilise alpha-amylase.
The use of pH sensors to monitor enzyme-catalysed decompositions has also been described in the literature. For example Ianniello and Yacynych, Anal. Chim. Acta (1983), 146, 249 report the use of an iridium dioxide-coated metal, for example titanium, as a pH responsive electrode for monitoring the decomposition of urea by urease. The urease was immobilised on to the iridium oxide by physical entrapment in a poly(vinyl chloride) film or by a covalent attachment via a cyanuric chloride linkage.
Fung et al, Analytical Chemistry, (1979) 51, 2319 describe a potentiometric enzyme electrode for the assay of methionine in solution. It was prepared by coating a layer of methionine lyase immobilised in bovine serum albumin cross-linked with glutaraldehyde on to an ammonia gas sensing electrode.
The non-covalent bonding of enzymes to a support surface is often a reversible process, and the equilibrium concentration of adsorbed enzyme may not be a useful amount. Moreover, enzyme which is adsorbed is frequently at least partially deaCtivated by distortion of its structure by bonding forces. The problem of deactivation by distortion can be particularly acute where regions of the enzyme are covalently bonded directly or through a bonding agent to functional groups on the surface of the support. This means that the desirable objective of immobilising an enzyme on a support without substantial loss of activity can only be met where there is a high degree of specificity between the enzyme, the support and the way in which they are linked.
In previously described biosensor devices comprising a sensor electrode in combination with an immobilised enzyme, the enzyme has been present as a dispersion in a relatively rigid film of polymeric material on the surface of the electrode or as a covalently-bonded coating on the surface of the electrode. Immobilisation in a rigid film imposes limitations on the accessibility of the substrate to the enzyme and on the reproducibility of such devices. The use of covalent bonding to immobilise the enzymes on the electrode surface restricts the amount of enzyme available in the system, while both methods of immobilisation limit the speed of response of the device.